FIG 2.

Intracellular B. abortus displays increased adhesion to, and internalization in, new host cells. HeLa cells grown on coverslips were infected with extracted intracellular B. abortus (Int) or in vitro-grown B. abortus (Ext) as indicated in Fig. 1 at a MOI of 10. At 2 h postinfection, cells were extensively washed, and living nonpermeabilized cells were incubated with an FITC-conjugated cow antibody against B. abortus for 30 min at 4°C to label extracellular bacteria (green). Cells were then fixed and permeabilized, and intracellular bacteria were further detected with a rabbit anti-Brucella antibody and Alexa Fluor Plus 594-conjugated goat anti-rabbit IgG (red). (A) Percentage of cells with associated bacteria; (B) mean number of bacteria per infected cell; (C) percentage of internalized Brucella; (D) number of intracellular bacteria in 100 cells. Each value is the average of at least three independent determinations. The statistical significance of differences from results for extracellular bacteria was calculated by Student’s t test (*, P < 0.05; **, P < 0.005).